Gene transfer-mediated angiogenesis therapy

ABSTRACT

The transgene-inserted replication-deficit adenovirus vector is effectively used in in vivo gene therapy for peripheral vascular disease and heart disease, including myocardial ischemia, by a single intra-femoral artery or intracoronary injection directly conducted deeply in the lumen of the one or both femoral or coronary arteries (or graft vessels) in an amount sufficient for transfecting cells in a desired region.

STATEMENT REGARDING FORMALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Nos.HL0281201 and HL1768218, awarded by the National Institutes of Health.The Government may have certain rights in this invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.08/396,207, filed on Feb. 28, 1995.

BACKGROUND

1. Field of the Invention

The present invention relates to gene therapy, more specifically, tovirus-mediated and other forms of gene therapy, and to certainadenovirus constructs useful in the delivery of desired genes. Moreparticularly, the invention relates to adenovirus-mediated delivery ofgenes useful in the promotion of angiogenesis in the heart, and tomethods for the treatment of peripheral vascular disease and diseases ofthe heart such as myocardial ischemia using such vectors.

2. Background of the Art

It has been reported by the American Heart Association (1995 StatisticalSupplement), that there are about 60 million adults in the United Statesthat have cardiovascular disease, including 11 million adults who havecoronary heart disease. Cardiovascular diseases are responsible foralmost a million deaths annually in the United States, representing over40% of all deaths. In 1995, 1.5 million adults in the United States willcarry the diagnosis of angina pectoris, experiencing transient periodsof myocardial ischemia resulting in chest pain. About 350,000 new casesof angina occur each year in the United States.

Myocardial ischemia occurs when the heart muscle does not receive anadequate blood supply and is thus deprived of necessary levels of oxygenand nutrients. The most common cause of myocardial ischemia isatherosclerosis, which causes blockages in the blood vessels (coronaryarteries) that provide blood flow to the heart muscle. Presenttreatments include pharmacological therapies, coronary artery bypasssurgery and percutaneous revascularization using techniques such asballoon angioplasty. Standard pharmacological therapy is predicated onstrategies that involve either increasing blood supply to the heartmuscle or decreasing the demand of the heart muscle for oxygen andnutrients. Increased blood supply to the myocardium is achieved byagents such as calcium channel blockers or nitroglycerin. These agentsare thought to increase the diameter of diseased arteries by causingrelaxation of the smooth muscle in the arterial walls. Decreased demandof the heart muscle for oxygen and nutrients is accomplished either byagents that decrease the hemodynamic load on the heart, such as arterialvasodilators, or those that decrease the contractile response of theheart to a given hemodynamic load, such as beta-adrenergic receptorantagonists. Surgical treatment of ischemic heart disease is based onthe bypass of diseased arterial segments with strategically placedbypass grafts (usually saphenous vein or internal mammary arterygrafts). Percutaneous revascularization is based on the use of cathetersto reduce the narrowing in diseased coronary arteries. All of thesestrategies are used to decrease the number of, or to eradicate, ischemicepisodes, but all have various limitations.

Preliminary reports describe new vessel development in the heart throughthe direct injection of angiogenic proteins or peptides to treatmyocardial ischemia. The several members of the fibroblast growth factor(FGF) family (namely acidic fibroblast growth factor, aFGF; basicfibroblast growth factor, bFGF; fibroblast growth factor-5, FGF-5 andothers) have been implicated in the regulation of angiogenesis duringgrowth and development. The role of aFGF protein in promotingangiogenesis in adult animals, for example, was the subject of a recentreport. It states that aFGF protein, within a collagen-coated matrix,placed in the peritoneal cavity of adult rats, resulted in a wellvascularized and normally perfused structure (Thompson, et al., PNAS86:7928-7932, 1989). Injection of bFGF protein into adult caninecoronary arteries during coronary occlusion reportedly led to decreasedmyocardial dysfunction, smaller myocardial infarctions, and increasedvascularity in the bed at risk (Yanagisawa-Miwa, et al., Science257:1401-1403, 1992). Similar results have been reported in animalmodels of myocardial ischemia using bFGF protein (Harada, et al., J ClinInvest 94:623-630, 1994, Unger, et al., Am J Physiol 266:H1588-H1595,1994).

A prerequisite for achieving an angiogenic effect with these proteinshowever, has been the need for repeated or long term delivery of theprotein, which limits the utility of using these proteins to stimulateangiogenesis in clinical settings. In other words, successful therapy inhumans would require sustained and long-term infusion of one or more ofthese angiogenic peptides or proteins, which are themselvesprohibitively expensive and which would need to be delivered bycatheters placed in the coronary arteries, further increasing theexpense and difficulty of treatment.

Recently, various publications have postulated on the uses of genetransfer for the treatment or prevention of disease, including heartdisease. See, for example, Mazur et al., "Coronary Restenosis and GeneTherapy," Molecular and Cellular Pharmacology, 21:104-111, 1994; French,"Gene Transfer and Cardiovascular Disorders," Herz 18:222-229, 1993;Williams, "Prospects for Gene Therapy of Ischemic Heart Disease,"American Journal of Medical Sciences 306:129-136, 1993; Schneider andFrench, "The Advent of Adenovirus: Gene Therapy for CardiovascularDisease," Circulation 88:1937-1942, 1993. Another publication, Leiden etal, International Patent Application Number PCT/US93/11133, entitled"Adenovirus-Mediated Gene Transfer to Cardiac and Vascular SmoothMuscle," reports on the use of adenovirus-mediated gene transfer for thepurpose of regulating function in cardiac vascular smooth muscle cells.Leiden et al. states that a recombinant adenovirus comprising a DNAsequence that encodes a gene product can be delivered to a cardiac orvascular smooth muscle cell and the cell maintained until that geneproduct is expressed. According to Leiden et al., muscle cell functionis regulated by altering the transcription of genes and changes in theproduction of a gene transcription product, such as a polynucleotide orpolypeptide. That polynucleotide or polypeptide, report Leiden et al.,interacts with the cardiac or smooth muscle cell to regulate function ofthat cell. Leiden et al. states that this regulation can be accomplishedwhether the cell is situated in vitro, in situ, or in vivo. Leiden etal. describes a gene transfer method comprising obtaining an adenoviralconstruct containing a gene product by co-transfecting a geneproduct-inserted replication deficient adenovirus type 5 (with the CMVpromoter) into 293 cells together with a plasmid carrying a completeadenovirus genome such as plasmid JMI7; propagating the resultingadenoviral construct in 293 cells; and delivering the adenoviralconstruct to cardiac muscle or vascular smooth muscle cells by directlyinjecting the vector into the cells.

There are impediments to successful gene transfer to the heart usingadenovirus vectors. For example, the insertion of a transgene into arapidly dividing cell population will result in substantially reducedduration of transgene expression. Examples of such cells includeendothelial cells, which make up the inner layer of all blood vessels,and fibroblasts which are dispersed throughout the heart. Targeting thetransgene so that only the desired cells will receive and express thetransgene, and the transgene will not be systemically distributed, arealso critically important considerations. If this is not accomplished,systemic expression of the transgene and problems attendant thereto willresult. For example, inflammatory infiltrates have been documented afteradenovirus-mediated gene transfer in liver (Yang, et al. Proc. Natl.Acad. Sci. (U.S.A.) 91:4407, 1994). Finally, with regard toadenovirus-mediated gene transfer of FGF-5 for the in vivo stimulationof angiogenesis, we have discovered that the injected viral material caninduce serious, often life-threatening cardiac arrhythmias.

The invention described and claimed herein addresses and overcomes theseand other problems associated with the prior art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic figure which shows rescue recombinationconstruction of a transgene encoding adenovirus.

FIG. 2 shows percent wall thickening (%WTh) in the ischemic bed duringright atrial pacing (HR=200 bpm), calculated by measuring end-diastolicwall thickness (EDWTh) and end-systolic wall thickness (ESVTh) beforeand 14±1 days after gene transfer with lacZ (control gene) and withFGF-5. Function in the ischemic bed was increased 2.6-fold aftertransfer with FGF-5 (p=0.0001) but unaffected by the control gene.

FIG. 3 shows the peak contrast ratio (a correlate of blood flow)expressed as the ratio of the peak video intensity in the ischemicregion (LCx bed) divided by the peak video intensity in theinterventricular septum (IVS), measured from the video images using acomputer-based video analysis program during atrial pacing (200 bpm)before and 14±1 days after gene transfer with lacZ (control gene) andwith FGF-5. Blood flow to the ischemic bed increased 2-fold normal aftergene transfer with FGF-5 (p=0.0018), but remained 50% of normal afterthe control gene.

FIG. 4A-4C show diagrams corresponding to myocardial contrastechocardiographs (not shown). White areas denote contrast enhancement(more blood flow) and dark areas denote decreased blood flow. FIG. 4Ashows acute LCx occlusion in a normal pig, FIG. 4B shows 14±1 days afterlacZ gene transfer, and FIG. 4C shows 14±1 days after gene transfer withFGF-5.

FIG. 5 shows the ratio of capillary number to fiber number quantitatedby microscopic analysis in the ischemic and nonischemic regions aftergene transfer with FGF-5 and with lacZ. There was increased angiogenesisafter FGF-5 gene transfer (p<0.038).

SUMMARY OF THE INVENTION

The present invention is directed to a gene therapy approach useful inthe treatment of heart disease, preferably myocardial ischemia, andperipheral vascular disease. One objective of the present invention isto provide a method for treating heart disease in which an angiogenicprotein or peptide, preferably FGF-5, is produced to a therapeuticallysignificant degree in the myocardium continuously for sustained periodsby targeting the heart with a vector construct containing a gene forsaid angiogenic protein or peptide, preferably a replication-deficientadenovirus construct, delivered through intracoronary injection,preferably by catheter introduced substantially (typically at leastabout 1 cm) beyond the ostium of one or both coronary arteries or one ormore saphenous vein or internal mammary artery grafts.

Another aspect of the present invention is a method for treating a heartdisease in a patient having myocardial ischemia, comprising delivering atransgene-inserted replication-deficient adenoviral vector to themyocardium of the patient by intracoronary injection, preferably asingle injection of the vector, directly into one or both coronaryarteries (or grafts), to transfect cardiac myocytes in the affectedmyocardium, said vector comprising a transgene coding for an angiogenicprotein or peptide such as FGF-5, aFGF, bFGF or VEGF (vascularendothelial growth factor), and expressing the transgene in the heart,thereby promoting angiogenesis in the affected region of the myocardium.By injecting the vector stock containing no wild-type virus deeply intothe lumen of one or both coronary arteries (or grafts), preferably intoboth the right and left coronary arteries (or grafts), and preferably inan amount of 10⁷ -10¹³ viral particles as determined by opticaldensitometry (more preferably 10⁹ -10¹¹ viral particles), it is possibleto locally transfect a desired number of cells, especially cardiacmyocytes, in the affected myocardium with angiogenic protein- orpeptide-encoding genes, thereby maximizing therapeutic efficacy of genetransfer, and minimizing undesirable angiogenesis at extracardiac sitesand the possibility of an inflammatory response to viral proteins. If aventricular myocyte-specific promoter is used, for example, the promotermore securely enables expression limited to the cardiac myocytes so asto avoid the potentially harmful effects of angiogenesis in non-cardiactissues such as the retina.

In another aspect, the present invention provides a filtered, injectableadenoviral vector preparation, comprising a recombinant adenoviralvector, preferably in a final viral titer of 10⁷ -10¹³ viral particles,said vector containing no wild-type virus and comprising a partialadenoviral sequence from which one or more required adenovirus genesconferring replication competence, for example, the E1A/E1B genes havebeen deleted, and a transgene coding for an angiogenic protein orpeptide such as angiogenic aFGF, bFGF and VEGF, driven by a promoterflanked by the partial adenoviral sequence; and a pharmaceuticallyacceptable carrier. By using this injectable adenoviral vectorpreparation, it is possible to perform effective adenovirus-mediatedFGF-5 gene transfer for the treatment of clinical myocardial ischemia orperipheral vascular disease without any undesirable effects.

In a further aspect, the present invention provides a method ofproduction of a viral stock containing a recombinant vector capable ofexpressing an angiogenic protein or peptide in vivo in the myocardium,comprising the steps of cloning a transgene, preferably coding for anangiogenic protein or peptide such as FGF-5, aFGF, bFGF and VEGF, into aplasmid containing a promoter and a polylinker flanked by partialadenoviral sequences of the left end of the human adenovirus 5 genomefrom which one or more required adenovirus genes conferring replicationcompetence, for example, the E1AJE1B genes have been deleted;co-transfecting said plasmid into mammalian cells transformed with themissing replication-requiring genes, with a plasmid which contains theentire human adenoviral 5 genome and an additional insert making theplasmid too large to be encapsidated, whereby rescue recombination takesplace between the transgene-inserted plasmid and the plasmid having theentire adenoviral genome so as to create a recombinant genome containingthe transgene without the replication-requiring genes, said recombinantgenome being sufficiently small to be encapsidated; identifyingsuccessful recombinants in cell cultures; propagating the resultingrecombinant in mammalian cells transformed with the absentreplication-requiring genes; and purifying the propagated recombinantsso as to contain the recombinant vector, without wild-type virustherein, and passing the purified vector through a filter, preferably10-50 micron filter, more preferably a 30 micron filter.

In yet another aspect, a recombinant adenovirus expressing an angiogenicpeptide or protein will be delivered by catheter into the proximalportion of the femoral artery or arteries, thereby effecting genetransfer into the cells of the skeletal muscles receiving blood flowfrom the femoral arteries. This will provide an angiogenic stimulus thatwill result in angiogenesis in skeletal muscle of the legs and willserve as a treatment for peripheral vascular disease, a disease that ischaracterized by insufficient blood supply to muscles of the legs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Transgenes Encoding Angiogenic Proteins and Peptides In the presentinvention, various protein or peptide growth factors that are capable ofimproving myocardial blood flow to ischemic regions of the heart (orskeletal muscle in the case of peripheral vascular disease) can be used.As an angiogenic protein or peptide to be expressed, an angiogenicprotein or peptide such as aFGF, bFGF, and FGF-5 can be exemplified. Theangiogenic activity of the FGF family is reasonably well established inthe setting of protein infusions (Yanagisawa-Miwa, et al., Science257:1401-1403, 1992, Harada, et al., J Clin Invest 94:623-630, 1994,Unger, et al., Am J Physiol 266:H1588-H1595, 1994). The gene for VEGF(vascular endothelial growth factor), a potent stimulater of new vesselgrowth, can also be used. Success of the gene transfer approach requiresboth synthesis of the gene product and secretion from the transfectedcell. From this point of view, a gene encoding FGF-5 is preferred and ispreferably selected to include a sequence encoding a signal peptide,thus directing that the gene product, once expressed, will gain accessto the cardiac (or skeletal muscle) interstitium and induceangiogenesis.

Helper Independent Replication Deficient Human Adenovirus 5 System

In general, the gene of interest is transferred to the heart (orskeletal muscle), including cardiac myocytes (and skeletal myocytes), invivo and directs constitutive production of the encoded protein. Severaldifferent gene transfer approaches are feasible. Preferred is thehelper-independent replication deficient human adenovirus 5 system.Using this system, we have demonstrated transfection greater than 60% ofmyocardial cells in vivo by a single intracoronary injection (Giordanoand Hammond, Clin. Res. 42: 123A, 1994). Non-replicative recombinantadenoviral vectors are particularly useful in transfecting coronaryendothelium and cardiac myocytes resulting in highly efficienttransfection after intracoronary injection. The same will be true fortransfecting desired cells of the peripheral vascular system.

The recombinant adenoviral vectors based on the human adenovirus 5(Virology 163:614-617, 1988) are missing essential early genes from theadenoviral genome (usually E1A/E1B), and are therefore unable toreplicate unless grown in permissive cell lines that provide the missinggene products in trans. In place of the missing adenoviral genomicsequences, a transgene of interest can be cloned and expressed intissue/cells infected with the replication deficient adenovirus.Although adenovirus-based gene transfer does not result in integrationof the transgene into the host genome (less than 0.1%adenovirus-mediated transfections result in transgene incorporation intohost DNA), and therefore is not stable, adenoviral vectors can bepropagated in high titer and transfect non-replicating cells. Althoughthe transgene is not passed to daughter cells, this is acceptable forgene transfer to adult skeletal muscle and cardiac myocytes, which donot divide. Retroviral vectors provide stable gene transfer, and hightiters are now obtainable via retrovirus pseudotyping (Burns, et al.,Proc Natl Acad Sci (USA) 90:8033-8037, 1993), but current retroviralvectors are unable to transduce nonreplicating cells (adult skeletalmuscle and cardiac myocytes) efficiently. In addition, the potentialhazards of transgene incorporation into host DNA are not warranted ifshort-term gene transfer is sufficient. Indeed, we have discovered thata limited duration expression of an angiogenic protein is sufficient forsubstantial angiogenesis, and transient gene transfer for cardiovasculardisease and peripheral disease processes is therapeutically adequate.

Human 293 cells, which are human embryonic kidney cells transformed withadenovirus E1A/E1B genes, typify useful permissive cell lines. However,other cell lines which allow replication-deficient adenoviral vectors topropagate therein can be used, including HeLa cells.

Construction of Recombinant Adenoviral Vectors

All adenoviral vectors used in the present invention can be constructedby the rescue recombination technique described in Graham, Virology163:614-617, 1988. Briefly, the transgene of interest is cloned into ashuttle vector that contains a promoter, polylinker and partial flankingadenoviral sequences from which E1A/E1B genes have been deleted. As theshuttle vector, plasmid pAC1 (Virology 163:614-617, 1988) (or an analog)which encodes portions of the left end of the human adenovirus 5 genome(Virology 163:614-617, 1988) minus the early protein encoding E1A andE1B sequences that are essential for viral replication, and plasmidACCMVPLPA (J Biol Chem 267:25129-25134, 1992) which contains polylinker,the CMV promoter and SV40 polyadenylation signal flanked by partialadenoviral sequences from which the EA/E1B genes have been deleted canbe exemplified. The use of plasmid PAC1 or ACCMVPLA facilitates thecloning process. The shuttle vector is then co-transfected with aplasmid which contains the entire human adenoviral 5 genome with alength too large to be encapsidated, into 293 cells. Co-transfection canbe conducted by calcium phosphate precipitation or lipofection(Biotechniques 15:868-872, 1993). Plasmid JM17 encodes the entire humanadenovirus 5 genome plus portions of the vector pBR322 including thegene for ampicillin resistance (4.3 kb). Although JM17 encodes all ofthe adenoviral proteins necessary to make mature viral particles, it istoo large to be encapsidated (40 kb versus 36 kb for wild type). In asmall subset of co-transfected cells, rescue recombination between thetransgene containing the shuttle vector such as plasmid pAC1 and theplasmid having the entire adenoviral 5 genome such as plasmid pJM17provides a recombinant genome that is deficient in the E1A/E1Bsequences, and that contains the transgene of interest but secondarilyloses the additional sequence such as the pBR322 sequences duringrecombination, thereby being small enough to be encapsidated (see FIG.1). With respect to the above method, we have reported successfulresults (Giordano, et al. Circulation 88:1-139. 1993, and Giordano andHammond, Clin Res 42:123A, 1994). The CMV driven β-galactosidaseencoding adenovirus HCMVSP1IacZ (CLIN RES 42:123A, 1994) can be used toevaluate efficiency of gene transfer using X-gal treatment.

The initial mode of gene transfer uses adenoviral vectors as delineatedabove. The advantages of these vectors include the ability to effecthigh efficiency gene transfer (more than 60% of target organ cellstransfected in vivo), the ease of obtaining high titer viral stocks andthe ability of these vectors to effect gene transfer into cells such ascardiac myocytes which do not divide.

Tissue-Specific Promoters

The present invention also contemplates the use of cell targeting notonly by delivery of the transgene into the coronary artery, or femoralartery, for example, but also the use of tissue-specific promoters. Byfusing, for example, tissue-specific transcriptional control sequencesof left ventricular myosin light chain-2 (MLC_(2V)) or myosin heavychain (MHC) to a transgene such as the FGF-5 gene within the adenoviralconstruct, transgene expression is limited to ventricular cardiacmyocytes. The efficacy of gene expression and degree of specificityprovided by MLC_(2V) and MHC promoters with lacZ have been determined,using the recombinant adenoviral system of the present invention.Cardiac-specific expression has been reported previously by Lee, et al.(J Biol Chem 267:15875-15885,1992). The MLC_(2V) promoter is comprisedof 250 bp, and fits easily within the adenoviral-5 packagingconstraints. The myosin heavy chain promoter, known to be a vigorouspromoter of transcription, provides a reasonable alternativecardiac-specific promoter and is comprised of less than 300 bp. Otherpromoters, such as the troponin-C promoter, while highly efficacious andsufficiently small, lacks adequate tissue specificity. By using theMLC_(2V) or MHC promoters and delivering the transgene in vivo, it isbelieved that the cardiac myocyte alone (that is without concomitantexpression in endothelial cells, smooth muscle cells, and fibroblastswithin the heart) will provide adequate expression of an angiogenicprotein such as FGF-5 to promote angiogenesis. Limiting expression tothe cardiac myocyte also has advantages regarding the utility of genetransfer for the treatment of clinical myocardial ischemia. By limitingexpression to the heart, one avoids the potentially harmful effect ofangiogenesis in non-cardiac tissues such as the retina. In addition, ofthe cells in the heart, the myocyte would likely provide the longesttransgene expression since the cells do not undergo rapid turnover;expression would not therefore be decreased by cell division and deathas would occur with endothelial cells. Endothelial-specific promotersare already available for this purpose (Lee, et al., J Biol Chem265:10446-10450, 1990).

In the present invention, with regard to the treatment of heart disease,targeting the heart by intracoronary injection with a high titer of thevector and transfecting all cell types is presently preferred.

Propagation and Purification of Adenovirus Vectors

Successful recombinant vectors can be plaque purified according tostandard methods. The resulting viral vectors are propagated on 293cells which provide E1A and E1B functions in trans to titers in thepreferred 10¹⁰ -10¹² viral particles/ml range. Cells can be infected at80% confluence and harvested 48 hours later. After 3 freeze-thaw cyclesthe cellular debris is pelleted by centrifugation and the virus purifiedby CsCl gradient ultracentrifugation (double CsCl gradientultracentrifugation is preferred). Prior to in vivo injection, the viralstocks are desalted by gel filtration through Sepharose columns such asG25 Sephadex. The product is then filtered through a 30 micron filter,thereby reducing deleterious effects of intracoronary injection ofunfiltered virus (life threatening cardiac arrhythmias) and promotingefficient gene transfer. The resulting viral stock has a final viraltiter in the range of 10¹⁰ -10¹² viral particles/ml. The recombinantadenovirus must be highly purified, with no wild-type (potentiallyreplicative) virus. Impure constructs can cause an intense immuneresponse in the host animal. From this point of view, propagation andpurification may be conducted to exclude contaminants and wild-typevirus by, for example, identifying successful recombinants with PCRusing appropriate primers, conducting two rounds of plaque purification,and double CsCl gradient ultracentrifugation. Additionally, we havefound that the problems associated with cardiac arrhythmias induced byadenovirus vector injection into patients can be avoided by filtrationof the recombinant adenovirus through an appropriately-sized filterprior to intracoronary injection. This strategy also appears tosubstantially improve gene transfer and expression.

Delivery of Recombinant Adenovirus Vectors

The viral stock can be in the form of an injectable preparationcontaining pharmaceutically acceptable carrier such as saline, forexample, as necessary. The final titer of the vector in the injectablepreparation is preferably in the range of 10⁷ -10¹³ viral particleswhich allows for effective gene transfer. Other pharmaceutical carriers,formulations and dosages are described below. The adenovirus transgeneconstructs are delivered to the myocardium by direct intracoronary (orgraft vessel) injection using standard percutaneous catheter basedmethods under fluoroscopic guidance, at an amount sufficient for thetransgene to be expressed to a degree which allows for highly effectivetherapy. The injection should be made deeply into the lumen (about 1 cmwithin the arterial lumen) of the coronary arteries (or graft vessel),and preferably be made in both coronary arteries, as the growth ofcollateral blood vessels is highly variable within individual patients.By injecting the material directly into the lumen of the coronary arteryby coronary catheters, it is possible to target the gene rathereffectively, and to minimize loss of the recombinant vectors to theproximal aorta during injection. We have found that gene expression whendelivered in this manner does not occur in hepatocytes and viral RNAcannot be found in the urine at any time after intracoronary injection.Any variety of coronary catheter, or a Stack perfusion catheter, forexample, can be used in the present invention. In addition, othertechniques known to those having ordinary skill in the art can be usedfor transfer of genes to the arterial wall.

For the treatment of peripheral vascular disease, a diseasecharacterized by insufficient blood supply to the legs, recombinantadenovirus expressing an angiogenic peptide or protein will be deliveredby a catheter that will be inserted into the proximal portion of thefemoral artery or arteries, thereby effecting gene transfer into thecells of the skeletal muscles receiving blood flow from the femoralarteries. This will provide an angiogenic stimulus that will result inangiogenesis in skeletal muscle of the legs.

Animal Model of Myocardial Ischemia

Important prerequisites for successful studies on gene therapy are (a)constitution of an animal model which is applicable to clinicalmyocardial ischemia which can provide useful data regarding mechanismsfor angiogenesis in the setting of myocardial ischemia, and (b) accurateevaluation of the effects of gene transfer. From this point of view,none of the prior art is satisfactory. We have made use of a porcinemodel of myocardial ischemia that mimics clinical coronary arterydisease. Placement of an ameroid constrictor around the left circumflex(LCx) coronary artery results in gradual complete closure (within 7 daysof placement) with minimal infarction (1 % of the left ventricle, 4±1%of the LCx bed) (Roth, et al. Circulation 82:1778, 1990, Roth, et al. AmJ Physiol 235:H1279, 1987, White, et al. Circ Res 71:1490, 1992,Hammond, et al. Cardiol 23:475, 1994, and Hammond, et al. J Clin Invest92:2644, 1993). Myocardial function and blood flow are normal at rest inthe region previously perfused by the occluded artery (referred to asthe ischemic region), due to collateral vessel development, but bloodflow reserve is insufficient to prevent ischemia when myocardial oxygendemands increase. Thus, the LCx bed is subject to episodic ischemia,analogous to clinical angina pectors. Collateral vessel development andflow-function relationships are stable within 21 days of ameroidplacement, and remain unchanged for four months (Roth, et al.Circulation 82:1778, 1990, Roth, et al. Am J Physiol 235:H1279, 1987,White, et al. Circ Res 71:1490, 1992). It has been documented bytelemetry that animals have period ischemic dysfunction in the bed atrisk throughout the day, related to abrupt increases in heart rateduring feeding, interruptions by personnel, etc. (unpublished data).Thus, the model has a bed with stable but inadequate collateral vessels,and is subject to periodic ischemia. Another distinct advantage of themodel is that there is a normally perfused and functioning region (theLAD bed) adjacent to an abnormally perfused and functioning region (theLCx bed), thereby offering a control bed within each animal.

Myocardial contrast echocardiography was used to estimate regionalmyocardial perfusion. The contrast material is composed ofmicroaggregates of galactose and increases the echogenicity (whiteness)of the image. The microaggregates distribute into the coronary arteriesand myocardial walls in a manner that is proportional to blood flow(Skyba, et al. Circulation 90:1513-1521, 1994). It has been shown thatpeak intensity of contrast is closely correlated with myocardial bloodflow as measured by microspheres (Skyba, et al. Circulation90:1513-1521, 1994). To document that the echocardiographic imagesemployed in the present invention were accurately identifying the LCxbed, and that myocardial contrast echocardiography could be used toevaluate myocardial blood flow, a hydraulic cuff occluder was placedaround the proximal LCx adjacent to the ameroid.

In the present study, when animals were sacrificed, the hearts wereperfusion-fixed (glutaraldehyde, physiological pressures, in situ) inorder to quantitate capillary growth by microscopy. PCR was used todetect angiogenic protein DNA and mRNA in myocardium from animals thathad received gene transfer. In addition, as described below, two weeksafter gene transfer, myocardial samples from all five lacZ-infectedanimals show substantial β-galactosidase activity on histologicalinspection. Finally, using a polyclonal antibody to an angiogenicprotein, angiogenic protein expression in cells and myocardium fromanimals that had received gene transfer was demonstrated.

The strategy for therapeutic studies included the timing of transgenedelivery, the route of administration of the transgene, and choice ofthe angiogenic gene. In the ameroid model of myocardial ischemia, genetransfer was performed after stable but insufficient collateral vesselshad developed. Previous studies using the ameroid model involveddelivery of angiogenic peptides during the closure of the ameroid, priorto the development of ischemia and collateral vessels. However, thisstrategy was not employed for several reasons. First, previous studiesare not suitable for closely duplicating the conditions that would bepresent in the treatment of clinical myocardial ischemia in which genetransfer would be given in the setting of ongoing myocardial ischemia;previous studies are analogous to providing the peptide in anticipationof ischemia, and are therefore less relevant. Second, it was presumed,based upon previous studies in cell culture, that an ischemic stimulusin conjunction with the peptide would be the optimal milieu for thestimulation of angiogenesis. This could optimally be achieved bydelivery of the transgene at a time when myocardial ischemia was alreadypresent. Linked to these decisions was the selection of the method toachieve transgene delivery. The constraint that the technique should beapplicable for the subsequent treatment of patients with coronarydisease, made several approaches untenable (continuous infusion of apeptide into the coronary artery, direct plasmid injection into theheart, coating the heart with a resin containing the peptide to providelong-term slow release). Finally, the pig model provided an excellentmeans to follow regional blood flow and function before and after genedelivery. The use of control animals that received the same recombinantadenovirus construct but with a reporter gene provided a control forthese studies. Those skilled in the art will understand that the resultsdescribed below in pigs are predictive of results in humans. The pig hasa native coronary circulation very similar of that of humans, includingthe absence of native coronary collateral vessels.

Therapeutic Applications

The replication deficient recombinant adenovirus vectors of the presentinvention allow for highly efficient gene transfer in vivo withoutcytopathic effect or inflammation in the areas of gene expression. Basedon these results, described further in the below Examples, it is seenthat a high enough degree of in vivo gene transfer to effect in vivofunctional changes is achieved.

In the case of treating a heart disease, the gene transfer of anangiogenic protein by intracoronary injection will promote angiogenesis.Thus, treatment of ischemia can be conducted after observation ofinitial ischemic episodes. In addition, after gene transfer, capillarynumber, blood flow and function will increase in the ischemic region.Application of these techniques clinically will be of great utility,especially initially in those with inoperative coronary artery diseaseand disabling angina pectoris. The data of the present inventiondemonstrate that gene transfer of a recombinant adenovirus expressingfibroblast growth factor-5 (FGF-5) is effective in substantiallyreducing myocardial ischemia.

Compositions or products of the invention may conveniently be providedin the form of formulations suitable for intracoronary administration. Asuitable administration format may best be determined by a medicalpractitioner for each patient individually. Suitable pharmaceuticallyacceptable carriers and their formulation are described in standardformulations treatises, e.g., Remington's Pharmaceuticals Sciences by E.W. Martin. See also Wang, Y. J. and Hanson, M. A. "Parental Formulationsof Proteins and Peptides: Stability and Stabilizers," Journals ofParental Sciences and Technology, Technical Report No. 10, Supp. 42:2S(1988). Vectors of the present invention should preferably be formulatedin solution at neutral pH, for example, about pH 6.5 to about pH 8.5,more preferably from about pH 7 to 8, with an excipient to bring thesolution to about isotonicity, for example, 4.5% mannitol or 0.9% sodiumchloride, pH buffered with art-known buffer solutions, such as sodiumphosphate, that are generally regarded as safe, together with anaccepted preservative such as metacresol 0.1% to 0.75%, more preferablyfrom 0.15% to 0.4% metacresol. The desired isotonicity may beaccomplished using sodium chloride or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycol,polyols (such as mannitol and sorbitol), or other inorganic or organicsolutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions. If desired, solutions of the above compositionsmay also be prepared to enhance shelf life and stability. Thetherapeutically useful compositions of the invention are prepared bymixing the ingredients following generally accepted procedures. Forexample, the selected components may be mixed to produce a concentratedmixture which may then be adjusted to the final concentration andviscosity by the addition of water and/or a buffer to control pH or anadditional solute to control tonicity.

For use by the physician, the compositions will be provided in dosageform containing an amount of a vector of the invention which will beeffective in one or multiple doses to induce angiogenesis at a selectedlevel. As will be recognized by those in the field, an effective amountof therapeutic agent will vary with many factors including the age andweight of the patient, the patient's physical condition, and the levelof angiogenesis to be obtained, and other factors.

The effective does of the compounds of this invention will typically bein the range of at least about 10⁷ viral particles, preferably about 10⁹viral particles, and more preferably about 10¹¹ viral particles. Thenumber of viral particles may, but preferably does not exceed 1013. Asnoted, the exact dose to be administered is determined by the attendingclinician, but is preferably in 1 ml phosphate buffered saline.

The presently preferred mode of administration in the case of heartdisease is by intracoronary injection to one or both coronary arteries(or to one or more saphenous vein or internal mammary artery grafts)using an appropriate coronary catheter. The presently preferred mode ofadministration in the case of peripheral vascular disease is byinjection into the proximal portion of the femoral artery or arteriesusing an appropriate arterial catheter.

To assist in understanding the present invention, the following Examplesare provided which describe the results of a series of experiments. Theexperiments relating to this invention should not, of course, beconstrued as specifically limiting the invention and such variations ofthe invention, now know or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the invention as described herein and hereinafter claimed.

EXAMPLE 1 Adenoviral Constructs

A helper independent replication deficient human adenovirus 5 system wasused. The genes of interest were lacZ and FGF-5. The full length cDNAfor human FGF-5 was released from plasmid pLTR122E (Zhen, et al. MolCell Biolo 8:3487, 1988) as a 1.1 kb ECOR1 fragment which includes 981bp of the open reading frame of the gene, and cloned into the polylinkerof plasmid ACCMVPLPA which contains the CMV promoter and SV40polyadenylation signal flanked by partial adenoviral sequences fromwhich the E1A and E1B genes (essential for viral replication) had beendeleted. This plasmid was co-transfected (lipofection) into 293 cellswith plasmid JM17 which contained the entire human adenoviral 5 genomewith an additional 4.3 kb insert making pJM17 too large to beencapsidated. Homologous rescue recombination resulted in adenoviralvectors containing the transgene in the absence of E1A/E1 B sequences.Although these recombinants were nonreplicative in mammalian cells, theycould propagate in 293 cells which had been transformed with E1A/E1B andprovided these essential gene products in trans. Transfected cells weremonitored for evidence of cytopathic effect which usually occurred 10-14days after transfection. To identify successful recombinants, cellsupernatant from plates showing a cytopathic effect was treated withproteinase K (50 mg/ml with 0.5% sodium dodecyl sulfate and 20 mM EDTA)at 56° C. for 60 minutes, phenol/chloroform extracted and ethanolprecipitated. Successful recombinants were then identified with PCRusing primers (Biotechniques 15:868-872, 1993) complementary to the CMVpromoter and SV40 polyadenylation sequences to amplify the insert (theexpected 1.1 kb fragment), and primers (Biotechniques 15:868-872, 1993)designed to concomitantly amplify adenoviral sequences. Successfulrecombinants then underwent two rounds of plaque purification. Viralstocks were propagated in 293 cells to titers ranging between 10¹⁰ and10¹² viral particles, and were purified by double CsCl gradientcentrifugation prior to use. Recombinant adenoviruses encodingβ-galactosidase, or FGF-5 were constructed using full length cDNAs. Thesystem used to generate recombinant adenoviruses imposed a packing limitof 5 kb for transgene inserts. The genes proposed, driven by the CMVpromoter and with the SV40 polyadenylation sequences were less than 4kb, well within the packaging constraints. Recombinant vectors wereplaque purified by standard procedures. The resulting viral vectors werepropagated on 293 cells to titers in the 10¹⁰ -10¹² viral particlesrange. Cells were infected at 80% confluence and harvested at 36-48hours. After freeze-thaw cycles the cellular debris was pelleted bystandard centrifugation and the virus further purified by double CsClgradient ultracentrifugation (discontinuous 1.33/1.45 CsCl gradient;cesium prepared in 5 mM Tris, 1 mM EDTA (pH 7.8); 90,000×g (2 hr),105,000×g (18 hr)). Prior to in vivo injection, the viral stocks weredesalted by gel filtration through Sepharose columns such as G25Sephadex. The resulting viral stock had a final viral titer in the 10¹⁰-10¹² viral particles range. The adenoviral construct was highlypurified, with no wild-type (potentially replicative) virus.

EXAMPLE 2 Adult Rat Cardiomyocytes in Cell Culture

Adult rat cardiomyocytes were prepared by Langendorf perfusion with acollagenase containing perfusate according to standard methods. Rodshaped cells were cultured on laminin coated plates and at 24 hours wereinfected with the β-galactosidase-encoding adenovirus obtained in theabove Example 1 at a multiplicity of infection of 1:1. After a further36 hour period the cells were fixed with glutaraldehyde and incubatedwith X-gal. Consistently 70-90% of adult myocytes expressed theβ-galactosidase transgene after infection with the recombinantadenovirus. At a multiplicity of infection of 1-2:1 there was nocytotoxicity observed.

EXAMPLE 3 Porcine Myocardium In Vivo

The β-galactosidase-encoding adenoviral vector obtained in Example 1 waspropagated in permissive 293 cells and purified by CsCl gradientultracentrifugation with a final viral titer of 1.5×10¹⁰ viralparticles, based on the procedures of Example 1. An anesthetized,ventilated 40 kg pig underwent thoracotomy. A 26 gauge butterfly needlewas inserted into the mid left anterior descending (LAD) coronary arteryand the vector (1.5×10¹⁰ viral particles) was injected in a 2 ml volume.The chest was closed and the animal allowed to recover. On the fourthpost-injection day the animal was killed. The heart fixed withglutaraldehyde, sectioned and incubated with X-gal for 16.5 hours. Afterimbedding and sectioning the tissue was counterstained with eosin.

Microscopic analysis of tissue sections (transmural sections of LAD bed96 hours after intracoronary injection of adenovirus containing lacZ)revealed a significant magnitude of gene transfer observed in the LADcoronary bed with many tissue sections demonstrating greater than 50-60%of the cells staining positively for β-galactosidase. Areas of themyocardium remote from the LAD circulatory bed did not demonstrate X-galstaining and served as a negative control, while diffuse expression of agene was observed in myocytes and in endothelial cells. The majority ofmyocytes showed β-galactosidase activity (blue stain), and, insubsequent studies using closed-chest intracoronary injection, similaractivity was present 14 days after gene transfer (n=8). There was noevidence of inflammation or necrosis in areas of gene expression

EXAMPLE 4 Porcine Ischemia Model

Animals included 18 domestic pigs (30-40 kg). A left thoracotomy wasperformed under sterile conditions for instrumentation. (Hammond, et al.J Clin Invest 92:2644-2652, and Roth, et al. J Clin Invest 91:939-949,1993). Catheters were placed in the left atrium and aorta, providing ameans to measure regional blood flow, and to monitor pressures. Wireswere sutured on the left atrium to permit ECG recording and atrialpacing. Finally, an ameroid was placed around the proximal LCx. After astable degree of ischemia had developed, the treatment group (n=11)received an adenoviral construct that included FGF-5 (an angiogenicgene), driven by a CMV promoter. Control animals (n=7) received genetransfer with an adenoviral construct that included a reporter gene,lacZ, driven by a CMV promoter.

Studies were initiated 35±3 days after ameroid placement, at a time whencollateral vessel development and pacing-induced dysfunction were stable(Roth, et al. Am J Physiol 253:H1279-1288, 1987, and Roth, et al.Circulation 82:1778-1789). Conscious animals were suspended in a slingand pressures from the LV, LA and aorta, and electrocardiogram wererecorded in digital format on-line (at rest and during atrial pacing at200 bpm). Two-dimensional and M-mode images were obtained using aHewleft Packard ultrasound imaging system. Images were obtained from aright parasternal approach at the mid-papillary muscle level andrecorded on VHS tape. Images were recorded with animals in a basal stateand again during right atrial pacing (HR=200 bpm). These studies wereperformed one day prior to gene transfer and repeated 14±1 days later.Rate-pressure products and left atrial pressures were similar in bothgroups before and after gene transfer, indicating similar myocardialoxygen demands and loading conditions. Echocardiographic measurementswere made using standardized criteria (Sahn, et al. Circulation 58:1072,1978). End-diastolic wall thickness (EDWTh) and end-systolic wallthickness (ESWTh) were measured from 5 continuous beats and averaged.Percent wall thickening (%VVTh) was calculated (EDWrh-ESWTh)/EDWTh!×100.Data were analyzed without knowledge of which gene the animals hadreceived. To demonstrate reproducibility of echocardiographicmeasurements, animals (n=5) were imaged on two consecutive days, showinghigh correlation (r² =0.90; p=0.005).

35±3 days after ameroid placement, well after ameroid closure, butbefore gene transfer, contrast echocardiographic studies were performedusing the contrast material (Levovist) which was injected into the leftatrium during atrial pacing (200 bpm). Studies were repeated 14±1 daysafter gene transfer. Peak contrast intensity was measured from the videoimages using a computer-based video analysis program (Color Vue II, NovaMicrosonics, Indianapolis, Ind.), that provided an objective measure ofvideo intensity. The contrast studies were analyzed without knowledge ofwhich gene the animals had received.

At completion of the study, animals were anesthetized and midlinethoracotomy performed. The brachycephalic artery was isolated, a canulainserted, and other great vessels ligated. The animals receivedintravenous heparin (10,000 IU) and papaverine (60 mg). Potassiumchloride was given to induce diastolic cardiac arrest, and the aortacross-clamped. Saline was delivered through the brachycephalic arterycannula (120 mmHg pressure), thereby perfusing the coronary arteries.Glutaraldehyde solution (6.25%, 0.1 M cacodylate buffer) was perfused(120 mmH pressure) until the heart was well fixed (10-15 min). The heartwas then removed, the beds identified using color-coded dyes injectedanterograde through the left anterior descending (LAD), left circumflex(LCx), and right coronary arteries. The ameroid was examined to confirmclosure. Samples taken from the normally perfused and ischemic regionswere divided into thirds and the endocardial and epicardial thirds wereplastic-imbedded. Microscopic analysis to quantitate capillary numberwas conducted as previously described (Mathieu-Costello, et al. Am JPhysiol 359:H204, 1990). Four 1 pm thick transverse sections were takenfrom each subsample (endocardium and epicardium of each region) andpoint-counting was used to determine capillary number per fiber numberratio at 400× magnification. Twenty to twenty-five high power fieldswere counted per subsample. Within each region, capillary number tofiber number rations were similar in endocardium and epicardium so the40-50 field per region were averaged to provide the transmural capillaryto fiber number ratio.

To establish that improved regional function and blood flow resultedfrom transgene expression, PCR and RT-PCR were used to detect transgenicFGF-5 DNA and mRNA in myocardium from animals that had received FGF-5gene transfer. Using a sense primer to the CMV promoterGCAGAGCTCGTTTAGTGMC! (SEQ ID NO.:1) and an antisense primer to theinternal FGF-5 sequence GAAAATGGGTAGAGATATGCT! (SEQ ID NO.:2), PCRamplified the expected 500 bp fragment. Using a sense primer to thebeginning of the FGF-5 sequence ATGAGCTTGTCCTTCCTCCTC! (SEQ ID NO.:3)and an antisense primer to the internal FGF-5 sequenceGAAAATGGGTAGAGATATGCT! (SEQ ID NO.:2), RT-PCR amplified the expected 400bp fragment.

Finally, using a polyclonal antibody directed against FGF-5 (Kitaoka, etal. Science 35:3189, 1994), FGF-5 protein expression was demonstrated 48hours as well as 14±1 days after FGF-5 gene transfer in cells andmyocardium from animals that had received gene transfer with FGF-5.

The helper independent replication deficient human adenovirus 5 systemconstructed in Example 1 was used to prepare transgene containingvectors. The genes of interest were lacZ and FGF-5. The materialinjected in vivo was highly purified and contained no wild-type(replication competent) adenovirus. Thus adenoviral infection andinflammatory infiltration in the heart were minimized. By injecting thematerial directly into the lumen of the coronary artery by coronarycatheters, it was possible to target the gene effectively. Whendelivered in this manner there was no transgene expression inhepatocytes, and viral RNA could not be found in the urine at any timeafter intracoronary injection.

Injection of the construct (4.0 ml containing about 10¹¹ viral particlesof adenovirus) was made by injecting 2.0 ml into both the left and rightcoronary arteries (collateral flow to the LCx bed appeared to come fromboth vessels). Animals were anesthetized, and arterial access acquiredvia the right carotid by cut-down; a 5F Cordis sheath was placed. A 5FMultipurpose (A2) coronary catheter was used to engage the coronaryarteries. Closure of the LCx ameroid was confirmed by contrast injectioninto the left main coronary artery. The catheter tip was then placed 1cm within the arterial lumen so that minimal material would be lost tothe proximal aorta during injection. This procedure was carried out foreach of the pigs.

Once gene transfer was performed, three strategies were used toestablish successful incorporation and expression of the gene. (1) Someconstructs included a reporter gene (lacZ); (2) myocardium from therelevant beds was sampled, and immunoblotting was performed toquantitate the presence of FGF-5; and (3) PCR was used to detect FGF-5mRNA and DNA.

The regional contractile function data in FIG. 2 shows that pigsreceiving lacZ showed a similar degree of pacing-induced dysfunction inthe ischemic region before and 14±1 days after gene transfer. Incontrast, pigs receiving FGF-5 gene transfer showed a 2.6 fold increasein wall thickening in the ischemic region during pacing (p=0.0001).These data demonstrate that FGF-5 gene transfer in accordance with theinvention was associated with improved contraction in the ischemicregion during pacing. Wall thickening in the normally perfused region(the interventricular septum) was normal during pacing and unaffected bygene transfer (% Wall Thickening: lacZ Group: Pre-gene, 56±11%,Post-gene, 51±9%; FGF-5 Group: Pre-gene, 63±7%, Post-gene, 58±5%; nodifferences, two-way analysis of variance). The data from the separatedeterminations were highly reproducible (lateral wall thickening: r²=0.90; p=0.005). The percent decrease in function measured bytransthoracic echocardiography was very similar to the percentagedecrease measured by sonomicrometry during atrial pacing in the samemodel (Hammond, et al. J Clin Invest 92:2644, 1993), documenting theaccuracy of echocardiography for the evaluation of ischemic dysfunction.Bars in FIG. 2 represent mean values, error bars denote 1 SE. FIGS.4A-4C are diagrams corresponding to myocardial contrastechocardiographs. FIG. 4A illustrates acute LCx occlusion in a normalpig, in which no flow is indicated in LCx bed (black) while septum (IVS)enhances (white), confirming that the image accurately identified theLCx bed and that reduced blood flow was associated with reduced contrastenhancement. FIG. 4B illustrates the difference in contrast enhancementbetween IVS and LCx bed 14 days after gene transfer with lacZ,indicating different blood flows in two regions during atrial pacing(200 bpm). In FIG. 4C, contrast enhancement appears equivalent in IVSand LCx bed 14 days after gene transfer with FGF-5, indicating similarblood flows in the two regions during atrial pacing.

FIG. 3 summarizes computer analysis of video intensity in the tworegions from all animals. In FIG. 3, data were expressed as the ratio ofthe peak video intensity (a correlate of myocardial blood flow) in theischemic region (LCx bed) divided by the peak video intensity in theinterventricular septum (IVS, a region receiving normal blood flowthrough the unoccluded left anterior descending coronary artery). Equalflows in the two regions would yield a ratio of 1.0. The ratio, prior togene transfer, averaged 0.5, indicates substantially less flow in theLCx bed that in the septum. FIG. 3 shows that animals receiving lacZgene transfer had a persistent blood flow deficit in the ischemicregion. Animals receiving FGF-5 gene transfer showed homogeneouscontrast enhancement in the two regions, indicating a 2-fold increase inmyocardial blood flow improved flow in the ischemic region (p=0.0018,two-way analysis of variance). Bars represent mean values, error barsdenote 1 SE.

The bar graph in FIG. 5 summarizes the microscopic analysis data,showing increased capillary number to fiber number ratio in the ischemicand nonischemic regions of animals that received gene transfer withFGF-5 when compared to the same regions of the hearts of animals thathad received gene transfer with lacZ. Bars represent mean values from5-6 animals in each group. Error bars denote 1 SE, p value from 2-wayanalysis of variance for gene effect. The analysis was performed withoutknowledge of treatment group.

Electropherograms upon the PCR amplification confirmed the presence ofJM17-CMV-FGF-5 DNA (the expected 500 bp fragment) in the LAD and LCxbeds of three pigs 14 days after gene transfer with FGF-5.Electropherogram upon the RT-PCR amplification confirmed the presence ofcardiac FGF-5 mRNA (the expected 400 bp fragment) in the LAD and LCxbeds 14 days after gene transfer with FGF-5, but not with lacZ. Inaddition, two weeks after gene transfer, myocardial samples from allfive lacZ-infected animals showed substantial β-galactosidase activityon histological inspection.

Finally, immunoblots of cell medium from cultured fibroblasts with theuse of the polyclonal antibodies to FGF-5 confirmed protein expressionand extracellular secretion 2 days after gene transfer of FGF-5 (n=4plates), but not after gene transfer of lacZ. Protein expression wasalso confirmed in myocardial samples 14±1 days after gene transfer ofFGF-5 but not after gene transfer of lacZ (n=4).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCAGAGCTCGTTTAGTGAAC20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAAAATGGGTAGAGATATGCT21                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGAGCTTGTCCTTCCTCCTC21                                                       __________________________________________________________________________

We claim:
 1. A method for stimulating coronary collateral vesseldevelopment in a patient, comprising delivering a replication-deficientadenovirus vector to the myocardium of the patient by intracoronaryinjection directly into one or both coronary arteries, said vectorcomprising a transgene coding for an angiogenic protein or peptide, andwhich expresses the transgene in the myocardium, thereby promotingcoronary collateral vessel development.
 2. The method of claim 1,wherein a single injection of said vector is delivered.
 3. The method ofclaim 1, wherein about 10⁷ to about 10¹³ adenovirus vector particles aredelivered in the injection.
 4. The method of claim 1, wherein about 10⁹to about 10¹² adenovirus vector particles are delivered in theinjection.
 5. The method of claim 1, wherein about 10¹¹ adenovirusvector particles are delivered in the injection.
 6. The method accordingto claim 1, wherein said transgene is driven by a CMV promoter which iscontained in the vector.
 7. The method according to claim 1, whereinsaid transgene is driven by a ventricular myocyte-specific promoterwhich is contained in the vector.
 8. The method according to claim 7,wherein said ventricular myocyte-specific promoter has the sequences ofventricular myosin light chain-2.
 9. The method according to claim 7,wherein said ventricular myocyte-specific promoter has the sequences ofmyosin heavy chain promoter.
 10. The method of claim 1, wherein saidangiogenic protein or peptide is a fibroblast growth factor.
 11. Themethod of claim 10, wherein said angiogenic protein is fibroblast growthfactor
 5. 12. The method of claim 10, wherein said angiogenic protein isacidic fibroblast growth factor.
 13. The method of claim 10, whereinsaid angiogenic protein is basic fibroblast growth factor.
 14. Themethod of claim 1, wherein said angiogenic protein is vascularendothelial growth factor.
 15. The method of claim 1, wherein saidintracoronary injection is conducted at least about 1 cm into the lumensof the left and right coronary arteries.
 16. The method of claim 1,wherein said intracoronary injection is conducted at least about 1 cminto the lumens of a sapheous vein graft and/or an internal mammaryartery graft in addition to a coronary artery.
 17. The method of claim1, wherein said transgene is operably linked to a tissue specificpromoter which is contained in the vector whereby expression of saidtransgene is controlled by said promoter.
 18. The method of claim 1,wherein said patient has myocardial ischemia.
 19. A method forstimulating vessel development in a patient having peripheral-deficientvascular disease, comprising delivering a replication-deficientadenovirus vector to the peripheral vascular system of the patient byintra-femoral artery injection directly into one or both femoralarteries, said vector comprising a transgene coding for an angiogenicprotein or peptide, and the transgene in the peripheral vascular system,thereby promoting peripheral vascular development.
 20. The method ofclaim 19, wherein a single injection of said vector is delivered. 21.The method of claim 19, wherein about 10⁹ to about 10¹³ adenovirusvector particles are delivered in the injection.
 22. The method of claim19, wherein about 10⁹ to about 10¹² adenovirus vector particles aredelivered in the injection.
 23. The method of claim 19, wherein about10¹¹ adenovirus vector particles are delivered in the injection.
 24. Themethod according to claim 33, wherein said angiogenic protein or peptideis selected from the group consisting of aFGF, bFGF, FGF-5, and VEGF.25. A method for treating heart myocardial ischemia, comprisingdelivering a replication-deficient adenovirus vector to the myocardiumof a patient by intracoronary injection, said vector comprising atransgene coding for an angiogenic protein or peptide, and whichexpresses the transgene in the myocardium, thereby promoting coronarycollateral vessel development.
 26. The method of claim 25, wherein saidintracoronary injection is directed to one or both coronary arteries.27. The method of claim 25, wherein said intracoronary injection isdirected to the left coronary artery.
 28. The method of claim 26,wherein said intracoronary injection is conducted at least about 1 cminto the lumens of both coronary arteries.
 29. The method of claim 28,wherein said intracoronary injection is conducted at least about 1 cminto the lumens of a saphenous vein graft or an internal mammary arterygraft in addition to the left or right coronary arteries.
 30. The methodof claim 29, wherein said intracoronary injection is conducted at leastabout 1 cm into the lumens of a saphenous vein graft or an internalmammary artery graft in addition to the left coronary artery.
 31. Themethod of claim 39, wherein said intracoronary injection is conducted atleast about 1 cm into the lumens of a saphenous vein graft or aninternal mammary artery graft in addition to the right coronary artery.32. The method of claim 25, wherein said angiogenic protein or peptideis a fibroblast growth factor.
 33. The method of claim 25, wherein saidangiogenic protein is fibroblast growth factor
 5. 34. The method ofclaim 25, wherein said intracoronary injection is directed to the rightcoronary artery.
 35. The method of claim 25, wherein said intracoronaryinjection is conducted at least about 1 cm into the lumen of the leftcoronary artery.
 36. The method of claim 25, wherein said intracoronaryinjection is conducted at least about 1 cm into the lumen of the rightcoronary artery.
 37. The method of any one of claims 25, wherein saidangiogenic protein or peptide is a vascular endothelial growth factor.38. The method of claims 1 or 25, wherein expression of said transgeneis limited to the heart.
 39. The method of claim 38, wherein expressionof said transgene is limited to the cardiac myocytes.
 40. The method ofclaim 39, wherein expression of said transgene is limited to theventricular cardiac myocytes.
 41. The method of claims 10 or 32, whereinsaid fibroblast growth factor is selected from the group consisting ofacidic fibroblast growth factor, basic fibroblast growth factor, andfibroblast growth factor 5.